Exploring the Plasmatic Platelet-Activating Factor Acetylhydrolase Activity in Patients with Anti-Phospholipid Antibodies

Autoimmun Highlights (2017) 8:5 https://doi.org/10.1007/s13317-017-0092-7

ORIGINAL ARTICLE

Exploring the plasmatic platelet-activating factor acetylhydrolase activity in patients with anti-phospholipid antibodies

1,2 1 1 2 Martina Fabris • Adriana Cifu` • Cinzia Pistis • Massimo Siega-Ducaton • 1 1,2 3 1,2 Desre` Ethel Fontana • Roberta Giacomello • Elio Tonutti • Francesco Curcio

Received: 23 December 2016 / Accepted: 12 March 2017 / Published online: 25 March 2017 Ó The Author(s) 2017. This article is an open access publication

Abstract Conclusions PAF-AH plasmatic activity is particularly up- Purpose To explore the role of plasmatic platelet-activat- regulated in LAC? and in ab2GPI IgG? patients, possibly ing factor acetylhydrolase (PAF-AH), a marker of cardio- representing an alternative prognostic biomarker for the vascular risk, in patients with anti-phospholipid antibodies therapeutic management of APS patients. (aPL). Methods PAF-AH activity was assessed in a series of 167 Keywords Platelet-activating factor acetylhydrolase Á unselected patients screened for aPL in a context of Anti-phospholipid syndrome Á Anti-beta2-glycoprotein I thrombotic events, risk of thrombosis or obstetric compli- antibodies Á Anti-prothrombin/phosphatidylserine cations and in 77 blood donors. antibodies Á Lupus anticoagulant Á Atherosclerosis Results 116/167 patients showed positive results for at least one aPL among IgG/IgM anti-prothrombin/phosphatidylserine (aPS/PT), anti-cardiolipin (aCL), anti-beta2- Introduction glycoprotein I (ab2GPI) or lupus anticoagulant (LAC), while 51/167 patients resulted aPL-negative. LAC? Anti-phospholipid syndrome (APS) is a hypercoagulable patients disclosed higher PAF-AH than LAC-negative disorder clinically displayed by venous or arterial throm- (22.1 ± 6.4 nmol/min/ml vs. 19.5 ± 4.1 nmol/min/ml; bosis and/or adverse obstetric events, accompanied by p = 0.0032), and aPL-negative patients (p = 0.03). persistent and elevated levels of anti-phospholipid anti- Patients presenting positive IgG ab2GPI disclosed higher bodies (aPL) [1]. According to the 2006 revised interna- PAF-AH than patients with only IgM ab2GPI-positive tional classification criteria [2], patients with definite antibodies (23.1 ± 7.2 nmol/min/ml vs. 20.1 ± 5.3 nmol/ diagnosis of APS are those presenting positive lupus anti- min/ml; p = 0.035), as well as than patients showing only coagulant (LAC) and/or one among anti-cardiolipin (aCL) isolated LAC, aCL or aPS/PT (16.9 ± 3.8 nmol/min/ml; IgG or IgM or anti-beta2 glycoprotein I (ab2GPI) IgG or p = 0.003). IgM antibodies. However, during the last international congress on aPL antibodies, the major experts defined the role of other so-called ‘‘non criteria’’ antibodies, con- & Martina Fabris [email protected] tributing to assess the risk of thrombosis or the identifica- tion of potential seronegative APS, such as the anti- 1 Department of Medical and Biological Sciences, University prothrombin/phosphatidylserine antibodies (aPS/PT) [3]. of Udine, Padiglione CSL - Via Chiusaforte, Ingresso F3, Of note, the combination of ab2GPI, aPS/PT and LAC has 33100 Udine, Italy 2 demonstrated the best diagnostic accuracy for APS [4] and Institute of Clinical Pathology, Department of Laboratory aPS/PT were recently recommended as a surrogate for Medicine, University Hospital of Udine, Padiglione CSL - Via Chiusaforte, Ingresso F3, 33100 Udine, Italy LAC when specific inhibitors and/or analytical variables may affect its interpretation [5]. However, no definite 3 Laboratory of Immunopathology and Allergy, Department of Laboratory Medicine, University Hospital of Udine, Udine, recommendations are available to guide the therapeutic Italy approach in patients positive only for aPS/PT antibodies. 123 5 Page 2 of 6 Autoimmun Highlights (2017) 8:5

Platelet-activating factor acetylhydrolase (PAF-AH) is a Methods family of enzymes, the most abundant of which is the plasma form, also called lipoprotein-associated phospho- The plasmatic PAF-AH activity was assessed by a colori- lipase A2 (Lp-PLA2) [6]. The plasmatic PAF-AH is con- metric assay (PAF-AH Assay Kit-Cayman Chemical stitutively active and circulates bound to LDL, HDL and Company, Ann Arbor, MI, USA). Briefly, plasma or serum other lipoproteins and catalyses the hydrolysis of the sn2 samples were incubated with the 2-thio PAF substrate, i.e., acetate of PAF and PAF mimetics, which are early medi- hydrolyzed by PAF-AH at the sn2-position releasing free ators of inflammation [7]. PAF activates a variety of cells thiols detected by DTNB Ellman’s reagent (5,50-dithio-bis- of the innate immune system promoting migration, adhe- 2-nitrobenzoic acid). sion and inflammatory effects. Thus, PAF-AH while Anti-cardiolipin (aCL) and anti-beta2 glycoprotein I inactivating PAF is considered an important factor in pre- (ab2GPI) IgG/IgM antibodies were investigated in all venting an exaggerated inflammatory response and in patients, while 125 patients were tested for lupus antico- protecting cells from uncontrolled oxidative damage [8]. agulant and 125 for anti-phosphatidylserine/prothrombin Several studies reported a significant association (aPS/PT) IgG/IgM antibodies. aCL and ab2GpI antibodies between higher PAF-AH plasmatic activity and the sever- were analysed by chemiluminescence (Zenit RA instru- ity of cardiovascular (CV) disease and identified PAF-AH ment by A. Menarini Diagnostics, Italy), while aPS/PT as a marker of vascular inflammation and atherosclerotic were assayed by Quanta Lite aPS/PT IgG/IgM ELISA kit plaque instability [9–11]. (Inova Diagnostics Inc, San Diego, CA). Plasma samples More and more papers in recent literature emphasize the were tested for the presence of LAC according to the relevant link between endothelial dysfunction, atheroscle- recommended criteria from the ISTH Subcommittee on rosis and APS [12–14]. Chronic inflammation is involved Lupus Anticoagulant-Phospholipid-dependent antibodies in various stages of development of the atherosclerotic and optimized according to recently published standard- plaques. Among the key molecules involved in the ization [17, 18]. Total cholesterol, low-density lipoproteins atherosclerotic process are heat-shock proteins, oxidized (LDL), high-density lipoproteins (HDL) and triglycerides LDL (oxLDL) and b2GPI. The latter is identified as an were analysed by diagnostic methods. anti-atherogenic agent involved in the atheromatous plaque formation in APS patients, since it is targeted by the Statistic analyses ab2GPI antibodies, typically associated with APS [15, 16]. In this study we analysed PAF-AH plasmatic activity in Quantitative variables were expressed as mean ± standard a large series of unselected patients screened for aPL deviation and checked for normality distribution by the antibodies in a reference laboratory for the diagnosis of Shapiro–Wilk test. Statistical analyses were performed autoimmune diseases, investigating its association with with GraphPad Prism software. To compare biomarker different pattern of aPL positivity. serum levels between patients and controls, either Mann– Whitney or unpaired t test was used when appropriate. Correlation analyses were performed using the Pearson’s or Materials and methods the Spearman’s rank correlation coefficient.

Patients Results The study was conducted in 167 consecutive unselected patients (124 females and 69 males; mean age: PAF-AH plasmatic activity in patients and controls: 51 ± 16 years) who were screened for the presence of aPL correlation with lipid metabolic markers at the Laboratory of Immunopathology of the University Hospital of Udine in the context of routine testing for PAF-AH plasmatic activity in BDs disclosed a mean value thrombotic events, risk of thrombosis or obstetric compli- of 15.6 ± 4 nmol/min/ml (range 5.9–28.4). As expected cations. Patients were compared to 77 blood donors (BDs; [11], a signiﬁcant correlation was found between PAF-AH 39 females and 38 males; mean age: 39 ± 13 years) and total cholesterol (r = 0.25; p = 0.032), a stronger enrolled at the Transfusion Unit of the same Hospital. All direct correlation with LDL (r = 0.46, p \ 0.0001) and a patients and controls gave their informed consent to the highly signiﬁcant inverse correlation with HDL study according to the Declaration of Helsinki (1964) and (r =-0.45, p \ 0.0001). No correlation was found with to the Italian legislation (Authorization of the Privacy age and sex (15.5 ± 5 nmol/min/ml in females vs. Guarantor No. 9, 12th of December 2013). 15.7 ± 3.3 nmol/min/ml in males).

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Of the 167 patients undergoing aPL investigation, 116 than LAC-negative patients (22.1 ± 6.4 nmol/min/ml vs. showed at least one positive aPL among LAC, aCL, ab2GPI 19.5 ± 4.1 nmol/min/ml; p = 0.0032). Of note, total or aPS/PT antibodies, while 51 resulted all negative. PAF- cholesterol levels did not differ between LAC? and LAC- AH plasmatic activity was markedly more elevated in the negative patients (202 ± 39 mg/dl vs. 201 ± 34 mg/dl; overall patients (19.8 ± 5.5 nmol/min/ml) than in BDs p = ns), as well as LDL (113 ± 39 mg/dl vs. (p \ 0.0001), but no difference was found between aPL? 108 ± 26 mg/dl; p = ns) and HDL serum levels and aPL-negative patients (19.9 ± 5.8 nmol/min/ml vs. (60 ± 21 mg/dl vs. 63 ± 21 mg/dl; p = ns). Moreover, 19.6 ± 4.7 nmol/min/ml; Fig. 1). LAC? patients disclosed higher PAF-AH than aPL-nega- Of note, total cholesterol levels did not differ signifi- tive patients (p = 0.03), with again no difference with cantly between BDs and the overall patients, nor between regard to HDL (62 ± 24 mg/dl in aPL-negative; p = ns) BDs and aPL? patients (188 ± 38 mg/dl vs. and LDL (127 ± 42 mg/dl in aPL-negative; p = ns). As 198 ± 42 mg/dl; p = 0.10) and between aPL? and aPL- illustrated in Fig. 2, patients presenting ab2GPI IgG? negative patients (206 ± 52 mg/dl; p = 0.47). However, antibodies disclosed higher PAF-AH plasmatic activity LDL serum levels were higher in aPL-negative patients than patients presenting only ab2GPI IgM? antibodies than in BDs (127 ± 42 mg/dl vs. 104 ± 35 mg/dl; (23.1 ± 7.2 nmol/min/ml vs. 20.1 ± 5.3 nmol/min/ml; p = 0.0073) as well as in aPL? patients (109 ± 35 mg/dl; p = 0.035), but they did not differ with regard to LDL and p = 0.032 vs. aPL-negative; p = ns vs. BDs). HDL serum levels. Patients who were negative for ab2GPI The significant correlation between PAF-AH activity and IgG or IgM antibodies, but who showed either isolated cholesterol, LDL and HDL serum levels persisted in aPL? LAC or aCL or aPS/PT-positive antibodies demonstrated patients (r = 0.21, p = 0.041; r = 0.23, p = 0.024 and significantly lower PAF-AH activities that appeared com- r =-0.31, p = 0.0027, respectively), while in aPL-negative parable to those measured in BDs (Fig. 2; patients it was evident only for LDL (r = 0.29, p = 0.14; 16.9 ± 3.8 nmol/min/ml; p = ns vs. BDs; p = 0.003 vs. r = 0.25, p = 0.0027 and r =-0.25, p = 0.21, respectively). ab2GPI IgM?). Total cholesterol, LDL and HDL serum levels in patients with isolated LAC or aCL or aPS/PT- PAF-AH plasmatic activity in patients disclosing positive antibodies did not differ from those measured in distinct pattern of aPL positivity patients with ab2GPI IgM? or IgG? antibodies. Overall, aPS/PT IgG? patients disclosed PAF-AH activity close to As shown in Fig. 1, when distinguishing aPL? patients that of aPS/PT IgM? patients (17.3 ± 3 nmol/min/ml vs. based on LAC assay, LAC? disclosed higher PAF-AH 16.1 ± 3.9 nmol/min/ml; p = ns). Finally, patients

Fig. 1 PAF-AH plasmatic activity in patients and controls. PAF-AH (19.9 ± 5.8 nmol/min/ml vs. 19.6 ± 4.7 nmol/min/ml; p = ns). plasmatic activity was markedly more elevated in the overall patients LAC-positive patients disclosed higher PAF-AH than LAC-negative (19.8 ± 5.5 nmol/min/ml) than in BDs (p \ 0.0001), but no differ- (22.1 ± 6.4 nmol/min/ml vs. 19.5 ± 4.1 nmol/min/ml; p = 0.0032) ence occurred between aPL-positive and aPL-negative patients 123 5 Page 4 of 6 Autoimmun Highlights (2017) 8:5

Fig. 2 PAF-AH plasmatic activity in patients with distinct aPL 20.1 ± 5.3 nmol/min/ml; p = 0.035). Patients negative for ab2GPI positivities. Patients presenting positive ab2GPI IgG antibodies IgG or IgM antibodies showing either isolated LAC or aCL or aPS/ disclosed higher PAF-AH plasmatic activity than patients presenting PT-positive antibodies (*) demonstrated significantly lower PAF-AH only positive ab2GPI IgM antibodies (23.1 ± 7.2 nmol/min/ml vs. activity (16.9 ± 3.8 nmol/min/ml; p = 0.003 vs. ab2GPI IgM?) disclosing ab2GPI IgG? antibodies together with aPS/PT Besides the presence of LAC, several different targets IgG? antibodies tended to show higher PAF-AH activity of aPL could be determined by a number of analytical than patients disclosing only ab2GPI IgG? antibodies methods, with frequent discordant results, that could (23.4 ± 7 nmol/min/ml vs. 21 ± 4.7 nmol/min/ml; make the laboratory diagnosis of APS extremely com- p = ns). plicated. The main role of the ab2GPI antibodies, espe- cially those specifically targeting domain I [20], is widely accepted and present results seem to further confirm their Discussion importance with regard to CV risk stratification, since PAF-AH appeared particularly elevated in ab2GPI-posi- Increased PAF-AH expression demonstrated a predictive tivepatientsandmoresointhose displaying LAC activity role for cardiovascular events in relation to the vulnera- and carrying the IgG isotype. This particular association bility of atherosclerotic plaques. Therefore, PAF-AH may be explained by the fact that IgG ab2GPI antibodies dosage has been proposed in the assessment of CV risk, to are able to recognize the stable complex between oxLDL ensure a better stratification of at risk populations [10]. To and b2GPI, thus facilitating macrophage-derived foam date, PAF-AH has never been investigated in the context of cell formation in patients with APS [15]. The immune- APS patients, or, even less, in patients at risk to develop an pathological mechanisms sustained by oxLDL/b2GPI overt APS (i.e. asymptomatic carriers of aPL antibodies, complexes are not yet fully understood, but Toll-like patients affected by systemic connective tissues diseases). receptor 4 (TLR4) was recently shown to be involved Our study was originally conducted in a context of [15]. TLR4 could be the key player linking PAF-AH up- patients routinely screened for APS, demonstrating a sig- regulationtoab2GPI IgG antibodies in APS, as evidenced nificant association between the presence of aPL antibod- by a mouse model of preterm delivery which demon- ies, LAC and ab2GPI IgG in particular, and PAF-AH up- strated that PAF effects and signalling depend upon TLR4 regulation in plasma. stimulation [21]. Atherosclerosis is definitely recognized as a chronic Lp-PLA2 activity proved to be markedly reduced inflammatory response to the accumulation of lipoproteins in vivo when the enzyme is bound to HDL [8], and this is in the walls of arteries [19]. PAF-AH, produced by in line with our observation that ab2GPI IgG? patients monocytes, macrophages and T lymphocytes, and mainly disclosed higher PAF-AH and lesser HDL than BDs. This associated with LDL, is predominantly expressed in the is not true for other subgroups of patients, such as aPL- necrotic centre of the atherosclerotic plaques and in the negative patients or those presenting only isolated LAC or macrophage-rich areas and releases pro-inflammatory aCL or aPS/PT antibodies. Compared to these patients, mediators, such as lysophospholipids and oxidized fatty PAF-AH plasmatic activity up-regulation in ab2GPI IgG? acids [8]. cases appeared to be at least partially disconnected from

123 Autoimmun Highlights (2017) 8:5 Page 5 of 6 5 the lipoprotein levels and specifically linked to the pres- antibodies appeared to be independent of common lipid ence of such aPL antibodies. metabolic markers (i.e. LDL), as previously reported by Therefore, PAF-AH up-regulation arose as a specific other authors in the context of patients with major coronary thrombotic risk marker in patients carrying ab2GPI anti- events [11]. The major international scientific societies of bodies and is not generally associated with other aPL cardiologists have included the measurement of PAF- antibodies possibly implicated in APS manifestations, but AH activity among the biomarkers useful in risk stratifi- further studies are needed to confirm this observation. cation of adult asymptomatic patients at intermediate car- Unfortunately, in two large randomized clinical trials, an diovascular risk in their guidelines [27]. inhibitor of PAF-AH (darapladib) [22, 23] failed to reduce Our data are encouraging, but since some cohort com- the risk of major coronary events as compared to placebo. parison include partially overlapping data, a definite utility In addition, it was associated with significantly higher rates of PAF-AH plasmatic activity as a new prognostic bio- of drug discontinuation and adverse effects. These results marker also in patients with aPL antibodies and/or definite suggested that PAF-AH may be a biomarker of vascular APS is actually precluded. For this reason further inflammation, rather than a causal pathway of CV diseases prospective studies on selected patients are ongoing. [23]. Therefore, high PAF-AH activity could reflect a response to pro-inflammatory stress characteristic both of Compliance with ethical standards atherosclerosis and APS [24]. Conflict of interest All authors declare no conflict of interest with The leading cause of death in primary and secondary APS regard to this paper. patients are cardiovascular events due to acceler- ated atherosclerosis, which often progresses more rapidly, Ethical approval All procedures performed in studies involving compared with the general population [14]. Some key pro- human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 inflammatory proteins correlate with APS clinical manifes- Helsinki declaration and its later amendments or comparable ethical tations [25] and common radiological markers of subclini- standards. cal atherosclerosis and CV risk were often reported in such patients [13]. However, to date, besides the presence of aPL Informed consent Informed consent was obtained from all individ- ual participants included in the study. itself, no serological biomarkers specifically associated with aPL-related pathogenic mechanisms have been identified as Open Access This article is distributed under the terms of the useful to improve the classification of CV risk in aPL? Creative Commons Attribution 4.0 International License (http://crea patients, with and without overt clinical manifestations. tivecommons.org/licenses/by/4.0/), which permits unrestricted use, In this scenario, present findings on PAF-AH assume a distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a relevant place, possibly representing a reliable and link to the Creative Commons license, and indicate if changes were affordable biomarker useful to identify patients at higher made. risk in which to take a more cautious therapeutic attitude in the follow-up. Moreover, studying PAF-AH metabolic pathway may References help to better explain the pathogenesis of APS and to improve management and interpretation of aPL-related 1. Khamashta M, Taraborelli M, Sciascia S, Tincani A (2016) Antiphospholipid syndrome. 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